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PubMed1991

Antibody-fluorescein conjugates for photoimmunodiagnosis of human colon carcinoma in nude mice.

To improve the detectability of tumors by light-induced fluorescence, the use of monoclonal antibodies (MoAb) as carriers of fluorescent molecules was studied. As a model for this approach, the biodistribution of an anticarcinoembryonic antigen (CEA) MoAb coupled to fluorescein was studied in mice bearing a human colon carcinoma xenograft. In vitro, such conjugates with fluorescein-MoAb molar ratios ranging from four to 19, doubly labeled with 125I, showed more than 82% binding to immobilized CEA. In vivo, conjugates with a fluorescein-MoAb molar ratio of ten or less resulted in a tumor uptake of more than 30% of the injected dose of radioactivity per gram tumor at 24 hours. Tumor to liver, kidney, and muscle ratios of 20, 30 and 72, respectively, were obtained 48 hours after injection of the 125I-MoAb-(fluorescein)10 conjugate. The highest fluorescence intensity was always obtained for the tumor with the anti-CEA MoAb conjugate; whereas in control mice injected with fluoresceinated control immunoglobulin G1, no detectable increase in tumor fluorescence was observed. To compare these results with a classically used dye, mice bearing the same xenografts received 60 micrograms of Photofrin II. The intensity of the fluorescence signal of the tumor with this amount of Photofrin II was eight times lower than that obtained after an injection of 442 ng of fluorescein coupled with 20 micrograms of MoAb, which gave an absolute amount of fluorescein localized in the tumor of up to 125 ng/g of tumor. These results illustrate the possibility of improving the specificity of in vivo tumor localization of dyes for laser-induced fluorescence photodetection and phototherapy by coupling them to MoAb directed against tumor markers.

Animals↗
PubMed1988

Enhancement of blood-brain barrier permeability to sodium fluorescein by stimulation of mu opioid receptors in mice.

The effects of opioids on the permeability of the blood-brain barrier (BBB) were examined in mice with sodium fluorescein as an indicator of the permeability. The brain was perfused with saline 30 min after injection of sodium fluorescein (40 mg/kg, i.v.) and examined by fluorometry. Morphine hydrochloride (0.3-10 mg/kg, s.c.) markedly increased the brain level of sodium fluorescein dose-dependently without influencing the plasma level, when administered 20 min before sodium fluorescein injection. Intracerebroventricularly (i.c.v.) injected morphine hydrochloride (0.5 and 1.0 microgram) increased the brain sodium fluorescein level. Buprenorphine (0.1 and 0.5 mg/kg, s.c.) was also effective. However, pentazocine, ethylketazocine, U-50488H and SKF-10047 had no significant influence. The i.c.v. administration of [D-Ala2, MePhe4, Gly(ol)5]enkephalin (0.1 microgram) and [D-Ala2, D-Leu5]enkephalin (0.5 microgram) but not of [D-Thr2, Leu5]enkephalin-Thr increased the brain level of sodium fluorescein significantly. A small dose of naloxone (i.p.) significantly inhibited the effects of morphine, buprenorphine, [D-Ala2, MePhe4, Gly(ol)5]enkephalin and [D-Ala2, D-Leu5]enkephalin. ICI-174864 coadministered i.c.v. with [D-Ala2, D-Leu5]enkephalin was ineffective in antagonizing the effect of the latter. These findings suggest that the stimulation of mu opioid receptors results in an increase in BBB permeability to sodium fluorescein.

Animals↗
PubMed1986

Aqueous to cornea fluorescein distribution ratio in normal and swollen cornea.

Intravitreal sodium fluorescein was used to simulate equilibrium fluorescein kinetics, thereby allowing simple measurement of the aqueous to cornea fluorescein distribution ratio. Two groups of rabbit corneas were studied: normal corneas and corneas wounded by freezing. The aqueous to cornea fluorescein distribution ratio was approximately 0.4, was not significantly different in groups of normal or wounded eyes and little variability was noted. In addition, a comparison of in vivo and in vitro measurements of corneal fluorescein concentration in wounded eyes suggests that in vivo protein-bound fluorescein in the cornea fluoresces less efficiently than free fluorescein.

Animals↗
PubMed1975

Histologic localization of sodium fluorescein in human ocular tissues.

We studied the distribution pattern of sodium fluorescein in human eyes microscopically. The ciliary body showed early and diffuse leakage, with staining of the basement membrane of the nonpigmented ciliary epithelium, indicating movement of fluorescein from the ciliary body into the aqueous humor. After five minutes the iris stroma stained with fluorescein, probably from the aqueous humor. The retinal vessels and retinal pigment epithelium were impermeable to fluorescein. Corresponding to the background fluorescence seen in fluorescein angiography, fluorescence was present in Bruch's membrane and in the stroma of the inner one third of the choroid. Drusen stained most intensely in areas of greatest PAS positively. Early fluorescence of the optic disk was the result of intravascular perfusion of the dye. Minimal diffusion of fluorescein from the fenestrated choroidal vessels across the border tissue of Elschnig into the peripheral optic nerve bundles was observed. Late fluorescene of the optic disk was due mainly to fluorescein staining of the lamina scleralis and glial columns.

Eye↗
PubMed1980

Indications for an oligomeric structure and for conformational changes in sarcoplasmic reticulum Ca2+-ATPase labelled selectively with fluorescein.

Fluorescein isothiocyanate is a highly specific inhibitor of the Ca2+-ATPase from sarcoplasmic reticulum. The Ca2+ pumping is inhibited completely at a fluorescein isothiocyanate concentration half that of the ATPase protein, indicating that the protein is at least a dimer. ATP protected specifically against fluorescein isothiocyanate inhibition, indicating that fluorescein isothiocyanate may react at the nucleotide binding site of the ATPase (probably with a reactive lysine residue). The fluorescein is incorporated almost exclusively into the 105 kdalton catalytic polypeptide of the ATPase and digestion by trypsin gives rise to a fluorescein-labelled 45 kdalton fragment. Conformational changes induced by addition of Ca can be studied conveniently with the fluorescein-labelled ATPase.

Animals↗
PubMed1983

Binding of fluorescein and carboxyfluorescein by human serum proteins: significance of kinetic and equilibrium parameters of association in ocular fluorometric studies.

The binding of fluorescein and 5- and 6-carboxyfluorescein by human serum proteins was measured at 37 and 4 degrees C by equilibrium dialysis. The equilibrium association constants (KA) for fluorescein were 3.7 X 10(3) and 7.1 X 10(3)M-1, and for carboxyfluorescein were 3.5 X 10(3) and 7.5 X 10(3)M-1 at 37 and 4 degrees C equilibrium dialysis data, was 3.9 X 10(3) binding sites in human serum, determined from the 37 degrees C equilibrium dialysis data, was 3.9 X 10(-3)M for fluorescein and 3.3 X 10(-3)M for carboxyfluorescein. Utilizing these binding parameters it was calculated that a maximum of 93.5% of the total fluorescein and 92.0% of the carboxyfluorescein would be bound by undiluted human serum proteins at 37 degrees C. Experimental binding data obtained after prolonged equilibrium dialysis (four days) at low total fluorochrome concentrations (1.5 X 10(-4)M or less) indicated that 93.1 +/- 1.0 (S.D.)% of the fluorescein and 90.1 +/- 0.7% of the carboxyfluorescein were bound at 37 degrees C by undiluted human serum proteins. Stopped-flow kinetic spectrophotometric studies of the changes in absorptivity at 487-488 and 510 nm that occurred when the fluorochromes were bound by human serum proteins, indicated that the fluorescein and carboxyfluorescein binding reactions were 99% complete within 0.65 and 1.72 sec. These had second-order association rate constants at 25 degrees C of 3.0 X 10(3) and 1.5 X 10(3)M-1 sec-1, respectively. These findings offer a basis for calculation of bound and free fluorescein and carboxyfluorescein in vivo in human subjects.

Binding Sites↗
PubMed1999

Oral fluorescein angiography with the confocal scanning laser ophthalmoscope.

OBJECTIVE: To evaluate the efficacy of oral fluorescein angiography with a confocal scanning laser ophthalmoscope (SLO) system. DESIGN: Comparative case series. PARTICIPANTS: The authors used a confocal SLO (Heidelberg Retina Angiograph [HRA]) to perform oral fluorescein angiography in 47 patients, 13 of whom were without any retinal disease and 34 with a variety of retinal diseases including macular holes and pucker, inflammatory diseases, retinal vascular diseases, and age-related macular degeneration. The images were also compared to images taken with a fundus camera after intravenous fluorescein injections in patients on whom both studies were done. INTERVENTION: Color fundus photographs were taken of each eye (30 degrees fundus camera) before drinking 4 ml of 25% sodium fluorescein mixed with 60 ml of orange juice. After oral fluorescein ingestion, images of each eye were taken with a fundus camera (TriX film) and the HRA (using 512- x 512-pixel resolution). The images were repeated at 0-, 2.5-, 5-, 7.5-, 10-, 12.5-, 15-, 20-, 25-, and 30-minute intervals. Twenty of the 47 patients underwent intravenous fluorescein angiography performed with the fundus camera. MAIN OUTCOME MEASURE: Images were analyzed by a masked reader, and foveal avascular zone visualization, branch retinal vessel identification, and image quality were scored. Statistical analysis was performed with a t test for paired data with a two-tailed test of significance (alpha = 0.05). RESULTS: Foveal avascular zone was 100% as seen in 16 eyes (47%) in the HRA machine versus 1 eye (2%) in the conventional fundus camera (P < 0.0001). The third-order branch retinal vessels were identified in 59% of eyes in the HRA versus 26% in the fundus camera group (P < 0.0001), and the image quality was considered comparable to an intravenous angiogram in 47% with the HRA versus 9% with the conventional fundus camera (P < 0.0001). CONCLUSIONS: Oral fluorescein angiography using the HRA produces sufficiently detailed images to diagnose, treat, and follow many types of retinal pathology.

Administration, Oral↗
PubMed1999

Fluorescein-ERG, a sensitive method for the detection of vascular damage in diabetic patients.

The purpose of this paper was to provide evidence for the reintroduction of simultaneously performed fluorescein angiography and electroretinography in the detection of diabetic retinopathy. ERG observations were made in conjunction with fluorescein angiography of 13 patients suffering from type I diabetes mellitus for five to 13 years. Only patients without any fluorescein leakage during angiography and without any morphologic changes in the fundus were involved in the study. Gold foil electrodes were used for recording. A stroboscopic lamp provided flashing light stimulation through a monochromatic blue filter. Intravenous fluorescein administration caused an immediate reduction in the ERG response. This reduction was seen both in the control subjects and in diabetes patients. In the control group, the reduction was over in 30-45 min, while in the diabetes group a considerable amplitude elevation was seen in all recordings between 15 and 60 min post-fluorescein. In the adaptation control group, where only repeated ERG recordings were employed every 15 min, a slight decrease in the a wave and a slight elevation of the b wave were observed during the whole recording period. No complaints or side-effects were detected during the observations. As all the patients displayed a normal fluorescein angiography besides elevated b wave after fluorescein administration, and this elevation was seen exclusively in the diabetic group, our study raises the possibility that this diagnostic method can be used in the detection of diabetic retinopathy.

Adolescent↗
PubMed1999

The effect of fluorescein on the accuracy of blood glucose determination by glucose meters.

PURPOSE: To investigate whether intravenous fluorescein significantly affected the accuracy of a glucose meter. METHODS: Thirty-four consecutive patients undergoing fluorescein angiography were recruited. Venous blood was removed immediately before and 1 hour after injection of 5 mls of 20% Sodium Fluorescein solution (1 mg). Whole blood glucose was measured with a glucose meter and plasma glucose levels were measured by the Department of Biochemistry for both samples. The results were analysed using Wilcoxon matched pairs test. RESULTS: Mean glucose results before fluorescein injection were 7.4 mmol/l (SD 4.5) with the glucose meter and 8.1 mmol/l (SD 5.0) biochemically (p=0.02). The post dose levels were 7.2 mmol/l (SD 4.7) and 8.5 mmol/l (SD 4.8), respectively (p=0.001). The difference between the mean results of the two tests was 0.7 mmol/l before fluorescein and 1.3 mmol/l after fluorescein (p=0.001) CONCLUSION: Intravenous fluorescein appears to influence minimally the difference between glucose levels measured in the laboratory and by glucose meter. The degree of difference, whilst achieving statistical significance, should not affect insulin dosage clinically.

Adult↗
PubMed1987

Fluorescein dilaurate serum test: a rapid tubeless pancreatic function test.

The value of a modified fluorescein dilaurate (FDL) serum test for the detection of pancreatic exocrine insufficiency was investigated in 89 patients with and without pancreatic disease. This test modification with fluorescein serum determination following metoclopramide (10 mg) and secretin (1 U/kg) i.v. injection appeared efficacious in a pilot study in six healthy volunteers. Individual peak fluorescein serum concentration was achieved within 180 min after the test meal in 96% of all subjects studied. Peak fluorescein serum concentration within this time period allowed the best discrimination between normal and abnormal pancreatic function. Sensitivity in detection of chronic pancreatitis was 86% (38 of 44 patients) when the lower normal fluorescein serum concentration was considered 4.5 micrograms/ml (this value corresponds to mean - 2 SD). The specificity of this test in detecting chronic pancreatitis was 100% when healthy controls were considered, but fell to 78% when patients with different gastrointestinal disorders, including those with secondary pancreatic insufficiency, were included. The correlation between serum and urinary fluorescein determination was significant (r = 0.61; p less than 0.01). Duodenal bicarbonate output/h after secretin also showed a significant correlation with peak fluorescein serum concentration (r = 0.79; p less than 0.001).

Adolescent↗
PubMed2005

Use of a high molecular weight fluorescein (fluorexon) ophthalmic strip in assessments of tear film break-up time in contact lens wearers and non-contact lens wearers.

PURPOSE: To assess whether fluorexon strips could be used in tear stability [tear film break-up time (TBUT)] measures, as compared with fluorescein strips. METHODS: The pre-ocular tear film stability was assessed in 40 adults (19-29 years of age), half of whom were adapted soft contact lens wearers, using the dye-assisted tear film break-up time (TBUT) method. The measures were carried out between 10.00 and 17.00 hours, with contact lens wearers removing their lenses just before the assessments. For all subjects, fluorescein or fluorexon ophthalmic strips (pre-wetted with preservative-free saline) were used to add dye to the tear film, the TBUT was assessed three times and then the eye lightly irrigated with saline. The measures were repeated 45 min later with the other dye, with half the subjects receiving fluorescein first and the other half receiving fluorexon first. RESULTS: Overall, the mean TBUT times were 12.2 +/- 5.9 s for fluorescein and 12.6 +/- 5.6 s for fluorexon (p = n.s.), but the non-lens wearers had longer TBUT times (14.6 and 14.5 s for the two dyes) compared with contact lens wearers (9.8 and 10.7 s, p < 0.01). While the inter-subject variability (as S.D.) was larger for non-contact lens wearers, the intra-subject variability [as coefficient of variation (COV)] was greater in the contact lens wearers when fluorescein was used (p < 0.001). When analysed as a function of the time of day that the measures were made, there were clear trends for both the fluorescein and fluorexon. TBUT values to increase in non-contact lens wearers and decrease in contact lens wearers. These trends failed to reach statistical significance, but the results with the contact lens wearers showed less variation than the non-contact lens wearers. CONCLUSION: The use of a fluorexon dye-impregnated strip (pre-wetted with preservative-free saline) yielded comparable results with a fluorescein strip for assessments of tear film stability based on TBUT. The studies also indicate that further research is needed on changes in TBUT values during the day.

Adult↗
PubMed1990

The effect of reduced tear drainage on corneal and aqueous concentrations of topically applied fluorescein.

The effect of reduced tear drainage on the intraocular penetration of topically applied fluorescein was studied with fluorophotometry in healthy eyes. Corneal and aqueous fluorescein concentrations could not be followed for the first hour after application of fluorescein because tear film fluorescense influenced fluorophotometric determinations of fluorescein concentrations in cornea and aqueous for at least 30 min. Insertion of punctal plugs in the upper and lower punctum of one eye caused a significant (P less than 0.025) increase in aqueous fluorescein concentrations 1 to 8 h after application of 20 microliters of a 2% solution of sodium fluorescein in the lower conjunctival sac. The achieved concentrations were almost 4 times as large as in the fellow eye. There was some individual variation, but a marked increase was observed in 9 of 11 subjects. Attempts to reduce tear drainage by compressing the tear sac and/or closing the eye lids for 1 min after application of the eye drop had no significant effect on corneal or aqueous concentrations of fluorescein 1 to 8 h later.

Administration, Topical↗
PubMed1982

Biliary secretion of sodium fluorescein in primary monolayer cultures of adult rat hepatocytes and its stimulation by nicotinamide.

Addition of sodium fluorescein to primary cultures of rat hepatocytes resulted in a rapid uptake of the dye by the hepatocytes and a subsequent accumulation in bile canaliculi-like structures. A similar distribution was obtained with fluorescein diacetate. Concentrations of Na-fluorescein accumulating within canaliculi varied over a wide range, often far exceeding that used in the medium. Ouabain strongly blocked cellular uptake, thus also impairing secretion of Na-fluorescein, whereas colchicine affected neither process. Taurolithocholate had virtually no influence on uptake, but markedly reduced the number of fluorescent canaliculi. Furthermore, fluorescent canaliculi could be discharged by addition of I M-sucrose in Hank's buffer, without affecting viability of the cultured cells. The percentage of canicular structures accumulating high amounts of Na-fluorescein markedly increased during cultivation for 7 days, concomitant with the progressive development of originally small and sporadic canaliculi into an anastomosing network of slender channels. This canalicular proliferation was strikingly reinforced by 20 mM-nicotinamide, resulting in an impressive network of canaliculi within 2-3 days. Nicotinamide also supported the secretion of Na-fluorescein, which could be stimulated further by addition of dehydrocholate. These results suggest that cultured hepatocytes are able to re-create a functional biliary polarity at least with respect to the biliary secretion of Na-fluorescein.

Animals↗
PubMed1975

Fluorescein diffusion in the human optic disc.

The characteristics of the transcapillary transfer of fluorescein dye in the optic disc of healthy individuals has been studied. A diffusible fluorescein dye and a nondiffusible reference substance, indocyanine green (ICG), which was assumed to remain in the capillaries, were injected into the circulatory system. The time courses of the concentrations of the two dyes in the optic disc were determined by simultaneously recording the fluorescence intensity of fluorescein and the infrared absorption by ICG with a fundus reflectometer. The difference between the fluorescein concentration curve and the reference ICG curve is a measure of the accumulation of fluorescein in the disc tissue. Our measurements indicate that fluorescein dye does not diffuse across the capillaries in the optic disc. The accumulation of fluorescein in the disc only starts at about one minute after the injection and seems to be due to diffusion of the dye from the surrounding choroid. The time constant of this diffusion process was found to be approximately one minute.

Adult↗
PubMed1980

Antihistamines as prophylaxis against side reactions to intravenous fluorescein.

Systemic antihistamines were administered prior to dye injection in 50 patients undergoing fluorescein angiography. The patients were monitored for side effects. Venous blood samples were obtained before and at three, ten and thirty minutes after intravenous administration of sodium fluorescein and analyzed for histamine levels. Three patients (6%) developed minor side effects of nausea or dizziness; this compares to an incidence of 21% in a previous series of patients from our institution untreated with antihistamines. A three-fold increase in plasma histamine levels occurred in 28% of patients following fluorescein and antihistamine injection; this compares with a 26% incidence of increase in plasma histamine in patients receiving fluorescein without antihistamines (as determined in a previous study). Prophylactic antihistamines should be considered in patients undergoing fluorescein angiography if they have a history of previous allergies or side reactions during prior fluorescein studies. However, complete prophylaxis against severe side reactions to fluorescein injections is not assured with antihistamines.

Adolescent↗
PubMed1994

Conversion of an anti-single-stranded DNA active site to an anti-fluorescein active site through heavy chain complementarity determining region transplantation.

Complementarity determining region (CDR) transplant studies were conducted between two monoclonal antibodies of distinctly different specificities (anti-fluorescein monoclonal antibody (mAb) 4-4-20 and anti-single-stranded DNA (ssDNA) mAb 04-01) which possessed nearly identical light chains but dissimilar heavy chains. The variations in binding specificities between the two immunoglobulins suggested that the active-site features of anti-fluorescein antibodies were dictated by characteristics intrinsic to the heavy chain (H-chain). To identify specific regions of the H-chain which influence the structure and function of an anti-fluorescein active site, CDR transplantation was systematically employed to convert the anti-ssDNA 04-01 antibody active site to an active site with anti-fluorescein activity. Each mAb 4-4-20 H-chain CDR (HCDR) was transplanted into the H-chain of a single-chain derivative of the 04-01 molecule. A fluorescence polarization ligand binding assay was utilized to determine the equilibrium dissociation constant, Kd, of hybrid transplant single-chain antibody HCDR1-HCDR2-HCDR3(4-4-20) for fluorescein (3.8 x 10(-7) M, indicating successful conversion of an anti-ssDNA active site to an anti-fluorescein binding site. A similar Kd (6.3 x 10(-7) M) was determined using a fluorescein fluorescence quenching assay. The transplantation results are discussed in terms of the relative contribution of each HCDR to a successful conversion in antibody specificity.

Amino Acid Sequence↗
PubMed1996

[Usefulness of the prick test for anaphylactoid reaction in intravenous fluorescein administration].

We examined whether a prick test was a valuable method in comparison with an intradermal skin test for predicting an anaphylactoid reaction to intravenous injection of fluorescein solution. Fifteen hundred cases were tested. The number (rate) of positive reactions to the prick test with 10% and 1.0% fluorescein solution was 2 (0.1%) and 0 (0.0%), respectively. In contrast, positive reaction to the intradermal skin tests with 10% and 0.1% fluorescein solution was observed in 686 cases (45.7%) and 13 cases (0.9%), respectively. Fluorescein angiography (FAG) was performed in 1,499 of the 1,500 cases. Adverse reactions such as nausea, cough, cold sweat, urticaria, and shock were noted in 85 cases (5.7%). Typical anaphylactoid shock occurred in one case (0.07%), which was one of the two cases positive to the prick test with 10% fluorescein. In the other positive prick test case, FAG was cancelled because of the high probability of anaphylactoid shock. The results suggest that a prick test with 10% fluorescein solution can markedly cut down the false positive reactions and can be a useful test for the prospective diagnosis of anaphylactoid reactions to intravenous fluorescein administration.

Adult↗
PubMed1998

The effect of sodium fluorescein angiography on erythrocyte properties.

Sodium fluorescein angiography is a widely used routine ophthalmological diagnostic procedure which enables the study of chorioretinal microcirculation and consists of the injection of sodium fluorescein into the systemic bloodstream. The aim of the present study was to evaluate whether or not fluorescein interferes with erythrocyte properties during the angiographic procedure. In a group of 37 patients, 26 with non-insulin-dependent diabetes mellitus (DM) with and without retinopathy, and 11 without diabetes mellitus (non-DM) although affected by other ophthalmological diseases, all undergoing routine angiography, blood samples were drawn before (T0) and 30 min (T30) after fluorescein injection. The erythrocyte aggregation index (EAI), membrane lipid fluidity and erythrocyte acetylcholinesterase activity were determined in both groups. After fluorescein injection there was no statistical change in EAI and erythrocyte membrane fluidity in either group. Erythrocyte acetylcholinesterase activity, a marker of membrane protein integrity, decreased significantly (p < 0.01) in the DM group. Membrane lipid fluidity did not change with fluorescein injection, however (i) in the DM group erythrocyte membranes became more rigid than in the non-DM (DPH: p < 0.01); (ii) EAI and membrane lipid fluidity became significantly correlated (r = 0.6263, p < 0.05) in non-DM patients at T30. In conclusion, fluorescein administration for angiographic procedures seems to interact with erythrocyte membrane, namely, in diabetic patients, which may interfere with the blood flow in the microcirculation.

Acetylcholinesterase↗